Discussion
The concept that the distribution of WHY1 between the nuclei and
chloroplasts plays a key role in the regulation of plant development has
arisen in recent years (Ren et al., 2017; Lin e al., 2019). The
phosphorylation of the WHY1 protein favours partitioning to nuclei, a
process that increase with leaf age (Guan et al ., 2018). However,
little information is available on the distribution of WHY1 between the
nuclei and proplastids of developing leaves. The immunogold labelling
analyses reported here revealed that over 60% of the WHY1 protein in
the basal sections of the leaves was located in the nuclei (Fig. 2).
Hence, it is likely that WHY1 fulfils its functions in the nuclei of
developing leaves as well as the plastids. Chloroplast development is
delayed in the absence of WHY1, a process that has largely been ascribed
to the functions of WHY1 in chloroplasts (Prikryl, Watkins, Friso, van
Wijk, & Barkam, 2008; Kurpinska et al., 2019). However, WHY1 functions
as a transcriptional activator in the nucleus, binding to the AT-rich
region of the kinesin gene promoter to activate kinesin gene expression
(Xiong et al., 2009), and to the GTCAAT motif of
the S40 promoter (Krupinska et al., 2014), and to a
combination motif of GTNNNAAT and AT-rich motif of downstream target
genes, such as WRKY53 , WRKY33 , SPO11 ,
and PR1 to regulate leaf senescence and other processes inA. thaliana(Miaoet al.,2013; Renet al., 2017; Huang et al. , 2018). Our transcriptome
profiles of the three developmental regions in the W1-1 and W1-7 leaves
compared to the wild type leaves provide some insights into how the
developmental pattern of transcripts is changed in the absence of WHY1.
While most transcripts exhibited similar patterns of abundance in the
developmental profiles of all genotypes there were some clear exceptions
such as a GNC transcription factor regulating stomatal development,
greening and chloroplast development and NAC1, an auxin-regulated
senescence-associated transcription factor, two transcripts encoding ARF
transcriptions factors with functions in leaf morphogenesis and
development and two transcripts with similarity to Arabidopsis GLK1 that
encodes a transcription factor required for the expression of nuclear
encoded photosynthetic genes (Waters et al., 2009). Moreover, SAG12
transcripts were more abundant in all the sections of the W1-7 line than
the other genotypes. These differences are indicative of divergent
chloroplast development programmes in leaves deficient in the WHY1
protein.
The WHY1 protein binds to ERF-binding cis elements in the promoter
regions of genes such as ERF109 (REDOX RESPONSIVE TRANSCRIPTION FACTOR
1, RRTF1) (Miao et al., 2013. ERF109 is involved in plant stress
responses and participates in reactive oxygen species (ROS) signalling
and the regulation of developmental programs, such as
jasmonate-dependent initiation of lateral root development (Huanget al. , 2019). WHY proteins have previously been reported to be
involved in the regulation of shoot and root development. For example,
WHY2 was shown to be a major regulator of root apical meristem
development (McCoy et al., 2021). Similarly, the expression of WHY1
(nWHY1) in the nucleus of Arabidopsis why1 mutants led to changes
in the levels of transcripts associated with plant development during
early growth, whereas expression of WHY1 in plastids increased the
abundance of transcripts associated with salicylic acid synthesis (Lin
et al., 2020). The binding of WHY proteins to the PB element of the9-cis epoxycarotenoid dioxygenase (NCED)1 gene in cassava
activated expression leading to increased abscisic acid levels (Yan,et al., 2020). Hence, the presence of WHY proteins in the nucleus
clearly influences the expression of genes involved in the synthesis of
phytohormones that control plant growth and defence. The primary cause
of the delay in greening observed in the barley leaves lacking WHY1 may
therefore result from the absence of WHY1-dependent regulation of
nuclear gene expression.
The action WHY1 as a transcription factor in the nucleus also regulates
the expression of genes associated with photosynthesis and carbon
metabolism. For example, WHY1 binds to the promoter of the rbcS gene
that encodes the small subunit of the potato ribulose-1, 5-carboxylase,
oxygenase under cold stress (Zhuang et al. , 2020), while WHY2
binds to the promoters of the SWEET11/15 genes that encode
sucrose transporters, leading to the modulation of starch allocation and
silique development (Huang et al ., 2020). Here we report that the
absence of WHY1 has a significant impact on the levels of transcripts
encoding enzymes associated with the Calvin cycle, starch and sugar
metabolism, glycolysis, the TCA cycle and amino acid metabolism, many of
which were more abundant in WHY1-deficient leaves than the wild type.
However, transcripts encoding enzymes such as β-amylase were less
abundant in WHY1 knockdown lines. WHY1 is known to bind to the ERE-like
element of the AMY3-L promoter, activating the expression of
amylase and starch degradation. WHY1 also binds to the ERE element of
the ISA2 promoter to inhibit isoamylase-mediated starch-synthesis
(Zhuang et al. , 2019). The absence of WHY1 from the nuclei of
developing barley leaves could therefore lead to the observed changes in
primary metabolites reported here. For example, all the metabolites of
the TCA cycle that were detected were significantly lower in WHY1
knockdown leaves than the wild type, as were GABA. Other amino acids
such as glycine, valine, leucine, threonine and isoleucine were higher
in WHY1-deficient leaves than the wild type. It may be that WHY1 can
bind to promotors of a wide range of housekeeping genes in the nucleus,
to modulate their expression in response to developmental and
environmental signals.
The WHY1-deficient barley leaves showed delayed chloroplast ribosome
formation and acquisition of photosynthetic activity suggesting that
WHY1 contributes to the coordination of nuclear and plastome gene
expression (Krupinska et al. , 2019). The role of the WHY proteins
in organelle to the nucleus retrograde signalling has long been
suspected (Foyer et al., 2014) but remains to be
established. Similarly, the
factors that determine the partitioning of WHY1 between the chloroplasts
and nuclei remain to be fully characterised. The compartmentation of
WHY1 between the plastids and nuclei is influenced by protein
phosphorylation, particularly as the leaves enter senescence (Renet al ., 2017). Phosphorylation of the WHY1 protein by CIPK14
kinase or oxidation by the addition of hydrogen peroxide causes a
re-distribution of WHY1 from the plastids to the nucleus (Ren et al.,
2017; Lin et al., 2019). However, little information is available about
the regulation of WHY1 partitioning in other situations such as the
bases of developing leaves. Earlier evidence indicated that WHY1 can
move from the plastids to the nucleus (Isemer et al., 2012). Direct
transfer of WHY1 from the plastids to the nuclei through contact sites
or stromules (Hanson et al, 2018) is possible but remains to be proven.
Moreover, the plastid-localized WHY1 affects miRNA biogenesis in the
nucleus (Swida-Barteczka et al., 2018) suggesting that WHY1 influences
chloroplast to nucleus signalling.
In summary, our understanding of WHY protein functions have greatly
increased in recent years, as has our knowledge of the flexibility of
their localisation and overlap of functions. The data presented here
provides new insights into the intracellular localization of the WHY1 in
developing leaves, highlighting how WHY1 in the nucleus might control
chloroplast development.